B vitamins for schizophrenia?

I'd like to briefly draw your attention to the results - systematic review and meta-analysis results - published by Joseph Firth and colleagues [1] observing that "certain vitamin and mineral supplements may reduce psychiatric symptoms in some people with schizophrenia" and specifically that certain B vitamins might be something to consider.

Such results come from a research team who are making significant waves in the field of meta-analyses and systematic reviews for all manner of different [important] topics. The additional inclusion of one Jerome Sarris to the authorship team adds a 'nutritional medicine as mainstream in psychiatry' touch to proceedings.

Drawing on data from 18 clinical trials - randomized controlled trials (RCTs) - cumulatively including over 800 participants, researchers reported that: "vitamin B supplementation (including B6, B8 and B12) reduced psychiatric symptoms significantly more than control conditions." Dose seemed to be important (higher doses appeared to be more effective than lower doses) as did timing of vitamin 'intervention'. Authors also indicated that subgroups of people with schizophrenia might be 'better responders' to this type of intervention, suggesting that either individual genetic differences or possibly nutritional deficiency before intervention might count in terms of effectiveness of B vitamin use. I was wondering whether those last points might tie into other discussions on this blog referencing genotype, B vitamins and [some] schizophrenia (see here).

In the context of the rise and rise of plurality in psychiatry ('the schizophrenias' and 'schizophrenia does not exist: discuss') there are some important research directions to be followed on the basis of the Firth findings. Identifying those people on the schizophrenia spectrum who might be potential best responders to this type of nutritional intervention is a research priority. Closely followed by further investigations on the hows-and-whys of such intervention potentially being useful. In that final respect, the peer-reviewed literature has already provided a few ideas for starters (see here and see here for examples).

To close, "I've got a good idea..."

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[1] Firth J. et al. The effects of vitamin and mineral supplementation on symptoms of schizophrenia: a systematic review and meta-analysis. Psychological Medicine. 2017. Feb 16.

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Firth J, Stubbs B, Sarris J, Rosenbaum S, Teasdale S, Berk M, & Yung AR (2017). The effects of vitamin and mineral supplementation on symptoms of schizophrenia: a systematic review and meta-analysis. Psychological medicine, 1-13 PMID: 28202095

ADHD and vitamin levels

"ADHD [attention-deficit hyperactivity disorder] patients were overrepresented in the group with low levels of some vitamins, possibly indicative of inadequate dietary intake of these micronutrients in a subgroup of patients. It is important to identify these patients in dietary intervention trials of ADHD."

So said the study findings reported by Elisabeth Toverud Landaas and colleagues [1] (open-access) providing some potentially important data on how nutritional factors might intersect with the diagnosis of ADHD. So: "Owing to the important and neurologically relevant functions of vitamins and the lack of studies exploring this topic in ADHD, we measured serum levels of the major vitamin classes in a sample of adult ADHD patients and controls to determine whether vitamin levels are associated with ADHD diagnosis and psychiatric symptoms." Said participants (n=133) were young adults and most were listed as having ADHD according to a "Norwegian national registry of adult ADHD patients." Vitamin levels were assessed from blood samples and compared with results for 131 control participants as per other studies on this cohort from this authorship group [2]. It's worth pointing out that samples were in deep freeze storage for between 2-9 years between collection and thawing for analysis.

Various vitamins were measured in those samples (vitamins A, B6, , B9, B12 and D to name a few) alongside levels of cotinine "to assess [tobacco] smoking status." The analytical assay(s) of choice was, in the most part, a familiar one to this blog: liquid- or gas chromatography-tandem mass spectrometry.

Results: "The concentrations of vitamins B2, B6 and B9 were all significantly lower in the ADHD group." When results were analysed according to percentiles based on blood levels of the various vitamins results similarly showed that those with ADHD were 'over-represented' in the lower levels bandings of those previously described vitamins. Smokers, as defined by a "widely used cut-off of 80 nmol/L" of blood cotinine, were also over-represented in the ADHD group (66%) compared with control participants' samples (12%). The authors reported that: "vitamin B6 and B9 levels were significantly higher in non-smoking ADHD patients compared with smokers" suggesting that lifestyle choices may play a role in some of the results obtained. Finally, when it came to looking at any possible association(s) between measured vitamin levels and behaviours pertinent to ADHD (derived from responses to the Adult ADHD Self-report Scale (ASRS), the authors report some preliminary observations but I'd like to see a little more data before anything further is made of this.

These are interesting results (aren't they always!). I note that the authors make reference to the findings reported by Julia Rucklidge and colleagues on a vitamin-mineral mix for ADHD (see here) and the idea that correcting vitamin deficiencies might have effects beyond just the somatic. There are however caveats to the latest results: "The reason why we observed association between lower levels of some vitamins and ADHD is uncertain and probably multifactorial. Regrettably, we do not have information on lifestyle and nutrient intake from the participants to help in the interpretation of our observations. It is reasonable to think that differences in dietary factors may partly be responsible for the differences." Indeed.

There is also one final observation to touch upon in the Landaas results concerning the vitamin/hormone of the hour: vitamin D. Although there was no overall difference in vitamin D concentrations in the ADHD and not-ADHD group samples, the authors did observe that: "for vitamin D, ADHD patients were significantly overrepresented both in the lowest and highest 10th percentile groups." Bearing in mind past research has suggested that ADHD might be yet another diagnosis/label where vitamin D deficiency might be a feature (see here) it is pertinent that the authors suggest: "One reason for the overabundance of ADHD patients in the highest 10th percentiles of vitamin D may thus be that relatively more ADHD patients take vitamin D supplements, either as part of an experimental treatment of symptoms or as a consequence of a diagnosed vitamin D deficiency."

Finally: "It is possible that low levels of certain vitamins may contribute to ADHD symptoms. Dietary intervention trials have shown promising effects in ADHD. Thus, identification and correction of low vitamin levels could be beneficial in treatment of ADHD. Further studies are warranted for replication and for examination of the underlying mechanisms."

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[1] Landaas ET. et al. Vitamin levels in adults with ADHD. BJPsych Open. 2016 Dec 13;2(6):377-384.

[2] Aarsland TI. et al. Serum concentrations of kynurenines in adult patients with attention-deficit hyperactivity disorder (ADHD): a case-control study. Behav Brain Funct. 2015 Nov 5;11(1):36.

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Landaas ET, Aarsland TI, Ulvik A, Halmøy A, Ueland PM, & Haavik J (2016). Vitamin levels in adults with ADHD. BJPsych open, 2 (6), 377-384 PMID: 27990293

Folate, autism and IMFAR: keep calm

IMFAR - The International Meeting for Autism Research - has just closed it's doors, bringing to an end one of the premier annual autism research conferences where one might expect quite a few of the presentations to eventually become (peer-reviewed) fodder for this blog.

This year (2016) has seen yet another startling array of research papers covering all manner of topics pertinent to autism; it's presumed aetiologies (plural as per the 'autisms'), diagnosis and management, all discussed and dissected. This year also saw some well needed focus on other important issues linked to autism such as various (medical and psychiatric) comorbidity linked to the label, the idea that children with autism become autistic adults (mostly) and the rather distressing idea that suicide (ideation or actual) seems to be over-represented when a diagnosis is received. Without trying to portray too negative an image of the very wide autism spectrum, autism can in some cases be a life-limiting as well as a life-changing condition (see here and see here).

As an outsider looking in on proceedings (#IMFAR2016) I was particularly interested in a few discussions that were covered during the event. This included the keynote address delivered by Irva Hertz-Picciotto on how, despite various environmental agents being traditionally correlated with some 'types' of autism (rubella, valproic acid), there are still challenges in terms of putting environmental factors on a par with genetic issues for example. Indeed, one or two tweets about her presentation kinda summed up the stark lack of knowledge and expertise in this area; specifically how 'chemical' insults (being careful with that word) potentially linked to cases of autism or at least autistic traits, already pervade the peer-reviewed literature (see here for example). It seems we need to further organise how research is done in this area, where exposure and genetic fragility are taken into account alongside the idea of synergy when it comes to the chemical soup that we all live in [1]; similar to the idea that multiple genes might be involved in multiple autisms so multiple non-genetic exposures might also show a connection (might I also suggest a greater focus on subgroups on the 'autisms' spectrum too?)

One of the other 'environmental' factors that was raised at IMFAR 2016 was that of folate (folic acid) and the possibility of a connection between pregnancy levels of this stuff and some autism as per the paper by Ramkripa Raghavan and colleagues [2]. For those who might already be well-read in autism research, the idea that maternal levels of folate during pregnancy might have a bearing on risk of offspring autism is not a new one (see here). With sentiments not a million miles away from those proposed by the late David Barker and the 'foetal programming hypothesis', the collected research on folate availability/supplementation during pregnancy impacting on the developing child is still the topic of some discussion (see here and see here). What is clear is that much like the need for folate during pregnancy to reduce the risk of neural tube defects, there may also be additional developmental requirements for suitable levels when it comes to other outcomes too.

Rather interestingly however, the data from Raghavan et al was not all one way when it came to 'risk' of offspring autism and levels of folate and a related nutrient, vitamin B12. To quote from their un-peer-reviewed paper: "In this urban low-income minority birth cohort, we observed an elevated risk of ASD [autism spectrum disorder] associated with high maternal plasma folate levels (>59 nmol/L), which far exceeds the excess cutoff suggested by the WHO (>45.3 nmol/L). Excess maternal vitamin B12 (>600 pmol/L) was also shown to be associated with greater ASD risk in offspring.  The risk of ASD was highest if mothers had both excess in folate and B12 levels." As you might imagine, the accompanying press release that followed this un-peer-reviewed paper was snapped up by various media outlets with titles like: "Taking too many vitamins during pregnancy 'can treble children's risk of being autistic'". Lo and behold, we have yet another 'scare story'...

Accepting that this was un-peer-reviewed research and that inflated press releases seem to abound in the domain of science communication (see here) I was a little less ruffled by the data reported by Raghavan and colleagues. I can see why such findings might make great headlines - "The risk was greatest for those children whose mothers had both high plasma folate (>59 nmol/L) and vitamin B12 (>600 pmol/L) (HR [adjusted hazard ratio]: 17.59; p value: <0.001)" - but this is not the first time that such sentiments have been expressed with relation to autism. Indeed, on a previous post asking whether some of the data of pregnancy folate levels/supplementation and offspring autism risk might not be just all about deficiency (see here) I discussed some rather speculative ideas (albeit peer-reviewed ideas) about how there may be a balance to be struck between potentially too little and too much of a good thing.

'Scientists urge caution over 'alarmist' claim of link between pregnancy folate and autism' was a rather more restrained headline in relation to the Raghavan report. I was much happier with this headline and coverage that put into perspective the preliminary nature of the report (yes, un-peer-reviewed) and how: “There are many epidemiologically based associations made of this sort – increasingly so in autism at the moment." Indeed there are, but unfortunately the commentator goes on to say that "Without details of the analysis, or any theory of action this looks like low-grade evidence."

Actually there are 'theories of action' and they've been discussed quite a bit in the peer-reviewed literature in this area. Not least, the idea that folate and vitamin B12 are important compounds in something called the folate cycle which intersects with another set of important metabolic process: the methylation cycle (see here) and all that DNA methylation stuff. There are a number of possible 'issues' that might be autism-relevant in these biological cycles, not least related to something called MTHFR (see here) and some emerging data on folate receptor autoantibodies (see here). Issues with these systems could very much impact on how folate is used and whether high plasma folate for example, might not be just as the result of too much supplementation. As per what we know from data from more formal medicines, drug metabolism can be quite an individual thing.

I do also want to bring in a little more data about vitamin B12 and 'some' autism as potentially being relevant. Accepting that there has been very little data on maternal vitamin B12 levels and offspring autism, there is certainly quite a bit of data out there about 'issues' with vitamin B12 being tied into specific cases of autism. This year (2016) we've seen the results of placebo-controlled study on the use of methyl B12 for aspects of autism (see here) as well as a suggestion that decreased brain levels of vitamin B12 might link cases of autism and schizophrenia (see here). That severe vitamin B12 deficiency has been linked to cases of Heller's syndrome is also potentially important (see here) given the focus on 'regression'...

Cumulatively what the Raghavan and other data point to is a potentially complicated relationship between mother's nutrition during pregnancy and offspring outcomes. I don't say this to somehow hark back to the darker days of autism theory in terms of 'blame' but rather, alongside other lines of evidence, to point out that nutrition during the nine months that made us (and perhaps earlier) plays an important role in making us who we are. The way that said nutrition is metabolised is also likely to vary from person to person. Minus sweeping generalisations and inflated media headlines, a greater research focus on how that nutrition might impact on at least some autism is very much implied again keeping in mind all that individuality in terms of how the body 'processes' nutrition. Indeed, for those mums identified in the Raghavan data as showing high levels of folate and vitamin B12, in the spirit of scientific endeavour, I'd be asking 'why?' and what does it mean for other related markers such as 'the big H' (homocysteine) for example?

For now however, keep calm and carry on with Love, love, peace, peace (song starts after 1 minute).

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[1] Boggess A. et al. Mean serum-level of common organic pollutants is predictive of behavioral severity in children with autism spectrum disorders. Sci Rep. 2016 May 13;6:26185.

[2] Raghavan R. et al. Maternal Plasma Folate, Vitamin B12 Levels and Multivitamin Supplement during Pregnancy and Risk of Autism Spectrum Disorders in the Boston Birth Cohort. IMFAR 2016; 22533. [NOT PUBLISHED IN A PEER-REVIEWED JOURNAL]

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Folate receptor autoantibodies (FRAAs) and a 'type' of autism?

"This study suggests that FRAAs [folate receptor α (FRα) autoantibodies] are associated with specific physiological and behavioral characteristics in children with ASD [autism spectrum disorder] and provides support for the notion that these biomarkers may be useful for subgrouping children with ASD, especially with respect to targeted treatments."

So said the study findings published by Richard Frye and colleagues [1] (open-access) who continued a research theme looking at FRAAs and their manifestation in 'some' autism. If you've already clicked that link in the last paragraph, you should have something of a flavour for what FRAAs are and what has already been discussed with autism in mind. If you didn't, the long and short of it is FRAAs describing the possibility of issues with folate transport as noted in the condition cerebral folate deficiency (CFD) are also being reported alongside some autism. The subsequent use of folinic acid (a vitamer of folic acid) to compensate might be something to consider for at least some on the autism spectrum bearing in mind my not giving any medical or clinical advice. I might also direct readers to a previous post with a helpful graphic on how the folate cycle also links in to some other important metabolic processes (see here) which is particularly timely in light of more publications on the topic of 'MTHFR'. I'll come back to this shortly.

This time around, Dr Frye and colleagues set about looking at whether those with autism also with issues around FRAAs present with a 'specific type' of autism, also taking into account different types of FRAAs - blocking and blinding. Serum samples for 94 children diagnosed with an ASD were analysed for blocking and binding FRAAs. At the same time, various markers covering redox, methylation, immune function and vitamin status were also determined and various measures of behaviour examined.

Results: "Fifty seven percent of the participants were positive for either the blocking or binding FRAAs, with 17% positive for blocking FRAA and 51% positive for the binding FRAA; 11% were positive for both FRAAs." From a behavioural/psychometric perspective: "ASD children positive for the blocking FRAA demonstrated better communication on the Vineland Adaptive Behavior Scale, stereotyped behavior on the Aberrant Behavioral Checklist and mannerisms on the Social Responsiveness Scale." In other words, those children with evidence of FRAAs, particularly blocking FRAAs, seemed to "have less severe ASD symptoms." The authors make mention of the term 'optimal outcome' with regards to this group, which is interesting when you consider the status of this often contentious line of research (see here).

The results of the various biological assays employed showed some interesting results. So: "ASD children with the blocking FRAA appear to have a more favorable redox and inflammation profile with relatively better glutathione and CT [3-Chlorotyrosine] indices than FRAA blocking negative children." Further, although folate levels were not significantly different between the groups on the basis of the presence of blocking or binding FRAAs (or neither), levels of vitamin B12 did show some differences: "Children positive for the binding FRAA were found to have higher serum B12 levels as compared to those negative for binding FRAAs."

Appreciating that it is still early days when it comes to FRAAs and autism, this and other research is crying out for independent replication with some appropriate cautions that FRAAs are not seemingly just confined to a diagnosis of autism (see here). The idea that those with autism with a specific type of FRAA (blocking) might present with a more favourable ASD profile in terms of symptoms and also biochemistry invites the question of whether the presence of such biology might actually be 'beneficial' bearing in mind the limited participant numbers included in the Frye study. I know that this might sound at odds with the whole folate-autism link that has been built up over the years, but as I've said before on this blog, folate metabolism and autism is a mighty complicated topic (see here). Likewise, is the idea that the presence of binding FRAAs might be something to 'target' given their seemingly less favourable biological and behavioural profile.

I do have one or two other points to make before I leave you. First, although mention is made of serum levels of the various biological analytes under investigation, one should be mindful of how representative these values are across the body. High serum vitamin B12 does not necessarily mean high brain levels of vitamin B12 for example (and alongside vitamin B12 I would have liked to have seen some data on the compound that is methylmalonic acid). Second, although mention is made of "methylenetetrahydrofolate reductase" (MTHFR), it would be interesting to see how many of the group presented with genetic issues with the production of this enzyme in light of previous findings (see here) and onwards the nature of any connection with FRAAs and autism.

This is interesting work but lots more investigation is implied.

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[1] Frye RE. et al. Blocking and Binding Folate Receptor Alpha Autoantibodies Identify Novel Autism Spectrum Disorder Subgroups. Front. Neurosci. 2016. March 9.

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Frye, R., Delhey, L., Slattery, J., Tippett, M., Wynne, R., Rose, S., Kahler, S., Bennuri, S., Melnyk, S., Sequeira, J., & Quadros, E. (2016). Blocking and Binding Folate Receptor Alpha Autoantibodies Identify Novel Autism Spectrum Disorder Subgroups Frontiers in Neuroscience, 10 DOI: 10.3389/fnins.2016.00080

Methyl B12 for autism? Placebo-controlled results say maybe...

"Methyl B12 treatment improved clinician-rated symptoms of ASD [autism spectrum disorder] that were correlated with improvements in measures of methionine metabolism and cellular methylation capacity."

Those were the very encouraging results published by Robert Hendren and colleagues [1] who can now update their ClinicalTrials.gov study entry (see here). Building on the ideas that: "Children with autism spectrum disorder (ASD) have been reported to have reduced ability to methylate DNA and elevated markers of oxidative stress" (topics that have been covered on this blog before), researchers undertook a gold-standard trial - randomised, placebo-controlled - to ascertain the effect (if any) of "8 weeks of treatment with methyl B12 (75 μg/kg) or saline placebo every 3 days in a subcutaneous injection." The success of the treatment was measured by "the Clinical Global Impressions-Improvement (CGI-I) score" accompanied by "changes in the Aberrant Behavior Checklist (ABC) and the Social Responsiveness Scale (SRS)" scores. At the same time, researchers also looked at various biochemical parameters pertinent to "methionine methylation and antioxidant glutathione metabolism."

Based on the 50 children ("mean age 5.3 years") who completed the study, researchers reported a trend of improvement in autistic and related behaviours following the methyl B12 injections. Importantly, the primary outcome measure - the CGI-I scores - rated by clinicians, showed a trend of being "statistically significantly better (lower) in the methyl B12 group (2.4) than in the placebo group (3.1) (0.7 greater improvement in the methyl B12 group, 95% CI 1.2-0.2, p = 0.005)." Biological parameters also showed changes: "increases in plasma methionine (p = 0.05), decreases in S-adenosyl-l-homocysteine (SAH) (p = 0.007) and improvements in the ratio of S-adenosylmethionine (SAM) to SAH (p = 0.007), indicating an improvement in cellular methylation capacity" following the use of methyl B12 compared with placebo.

Accepting that 'subcutaneous injection' of methyl B12 is hardly a 'user-friendly' option and may very well scupper plans to use this particular intervention option for quite a few, these are potentially important results. I'm really quite interested in how vitamin B12 'vitamers' might show some links to at least some 'types' of autism (see here) including the measurement of 'brain levels' of the stuff (see here). The Hendren results suggest that quite a few more research resources might be needed in this area. I wonder also if this future research agenda would include the 'baby and bathwater' compound that is methylmalonic acid in relation to autism too (see here)?

I do also have to point out that previous research from members of this research team has not been so complimentary about the use of methyl B12 in cases of autism [2] despite the idea that there may be 'responders' to this type of intervention. To quote: "methyl B12 may alleviate symptoms of autism in a subgroup of children, possibly by reducing oxidative stress. An increase in glutathione redox status (GSH/GSSG) may provide a biomarker for treatment response to methyl B12." Such differences in reported results are not unfamiliar to autism research (the rule rather than the exception) but perhaps provides a further focus for clarification of effect.

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[1] Hendren RL. et al. Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. J Child Adolesc Psychopharmacol. 2016 Feb 18.

[2] Bertoglio K. et al. Pilot study of the effect of methyl B12 treatment on behavioral and biomarker measures in children with autism. J Altern Complement Med. 2010 May;16(5):555-60.

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Hendren RL, James SJ, Widjaja F, Lawton B, Rosenblatt A, & Bent S (2016). Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. Journal of child and adolescent psychopharmacology PMID: 26889605

Decreased brain levels of vitamin B12 in autism

I have to thank Dr Malav Trivedi for bringing my attention to some recent findings reported by Yiting Zhang and colleagues (including Malav) [1] (open-access) suggesting that: "levels of vitamin B12, especially its MeCbl [methylcobalamin] form, decrease with age in frontal cortex of control human subjects."

Further, researchers reported: "abnormally lower total Cbl [cobalamin] and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls." Some media on the findings can also be read here.

Working from the lab of Dr Richard Deth (quite a familiar name to this blog), researchers initially analysed a most precious sample medium (postmortem brain samples) obtained from various biobanks and including various patient groups. So alongside samples from 12 children with autism were samples from 9 people diagnosed with schizophrenia and some 43 'controls' with ages ranging between 19 weeks old and 80 years old. "Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites" accompanied by data derived from a knock-out mouse model: "the influence of decreased GSH [glutathione] production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70%."

Looking at postmortem frontal cortex brain samples, researchers reported that finding on levels of vitamin B12 - particularly the MeCbl vitamer -  decreasing with age. Bearing in mind the relatively small participant numbers included, the idea that lower brain tissue levels of total cobalamin and methylcobalamin were also present (almost unanimously) in the autism and schizophrenia groups could be important. I might at this point direct readers to previous discussions on vitamin B12 and autism on this blog (see here) including the research idea of supplementing (see here) with no medical advice given or intended.

There are a few other details worth pointing out from the Zhang findings. Analysis of thiols in brain samples across the autism vs control group revealed some potentially interesting data. So, methionine levels were quite a bit lower in the autism group [significantly lower] as were levels of "the methyl donor S-adenosylmethionine (SAM)." Both these compounds form an important part of the whole 'methylation of DNA' process (see here) among other things.

Glutathione, a compound that has seen its fair share of speculation with autism in mind (see here), was also on the research menu in the Zhang study. Interestingly and again bearing mind the small participant numbers, brain levels of this stuff were lower in the autism group as a whole but not significantly so when compared to controls. This finding might map on to other brain studies with autism in mind (see here). Likewise, cysteine (another potentially relevant compound to some autism) produced a similar finding.

I would encourage readers to take some time looking at the Zhang paper. In conjunction with other results reporting on some important elements to the emerging story (see here) I believe there are further studies to be done applicable to the notion that: "impaired methylation may be a critical pathological component" for at least some autism (see here). Indeed, other research papers have also discussed this issue [2]. The idea that studies about human ageing may likewise be informative to autism (and schizophrenia) research also carries quite a lot of traction too.

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[1] Zhang Y. et al. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797.

[2] Keil KP. & Lein PJ. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders? Environmental Epigenetics. 2016; 1-15.

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Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, & Deth RC (2016). Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PloS one, 11 (1) PMID: 26799654

Maternal folate status and offspring autism risk: where are we up to?

I'd like to briefly draw your attention to the review published by Elizabeth DeVilbiss and colleagues [1] today, covering "what is known about the role of folate in the aetiology of neurodevelopmental disorders."

Folate, is a topic that has graced this blog a few times with autism in mind (see here for example) based on various ideas that folate status during pregnancy might have the ability to modify offspring risk of autism [2] alongside the idea that autoimmune processes might act on folate receptors in some cases of autism (see here) and what this might subsequently mean for pathology / management. The specific idea that folate levels and folate supplementation during pregnancy might influence autism risk has garnered the most research attention, seemingly also crossing geographies too [3].

The DeVilbiss review is quite comprehensive in its scope and material covered, summarising "relevant biological, genetic and epigenetic mechanisms" and the various science that has been done so far on this topic. I would certainly agree with their sentiments that "existing evidence is inconclusive" (as previously indicated) in light of the numerous confounding variables also potentially linked to offspring autism risk. That being said, and acknowledging where folate metabolism sits in terms of areas such as MTHFR genetics (see here) and the whole vitamin B12 story (see here) and perhaps beyond (see here), I do think there is more to see in this area and perhaps outside of autism and related neurodevelopmental conditions (see here). Without jumping on the whole epigenetics bandwagon, the link between the folate cycle and DNA methylation in particular (see here) offers a whole slew of research ideas ripe for further investigation.

Music: Lost Frequencies - Are You With Me.

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[1] DeVilbiss EA. et al. Maternal folate status as a risk factor for autism spectrum disorders: a review of existing evidence. Br J Nutr. 2015 Aug 5:1-10.

[2] Schmidt RJ. et al. Maternal periconceptional folic acid intake and risk of autism spectrum disorders and developmental delay in the CHARGE (CHildhood Autism Risks from Genetics and Environment) case-control study. Am J Clin Nutr. 2012 Jul;96(1):80-9.

[3] Surén P. et al. Association between maternal use of folic acid supplements and risk of autism spectrum disorders in children. JAMA. 2013 Feb 13;309(6):570-7.

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DeVilbiss EA, Gardner RM, Newschaffer CJ, & Lee BK (2015). Maternal folate status as a risk factor for autism spectrum disorders: a review of existing evidence. The British journal of nutrition, 1-10 PMID: 26243379

Can a gluten-free diet positively affect cognitive performance?

No, I am not suggesting that a gluten-free diet is the new nootropic (cognitive enhancer) of choice with the title of this post despite previous media headlines on the subject.

"Got gluten?"☺  @ Wikipedia 

I am however very, very interested in the results reported by Irene Lichtwark and colleagues [1] (open-access here) which suggested that in "newly diagnosed coeliac disease, cognitive performance improves with adherence to the gluten-free diet in parallel to mucosal healing".

For those who might not be familiar with coeliac (celiac) disease (CD), I wrote a sort of training post about what we (think we) know about the condition a few months back (see here). An autoimmune condition managed in the most part by the lifelong use of a gluten-free (GF) diet, CD remains the focal point of an increasing 'spectrum' of conditions where gluten might play some kind of role (see here) including those linked to brain and behaviour (see here). Indeed the Lichtwark paper starts with the assumption that outside of the classical genetic, gastrointestinal (GI) and immunological markers/effects of CD, there is a growing recognition that other biological systems may be affected, something I've talked about before on this blog.

The Lichtwark paper is open-access but as ever, here are a few pointers:

• A small participant group of 11 adults (mean age 30 years old) diagnosed with CD but not yet commencing with a GF diet were followed over the course of 1 year. Various assessments were periodically carried out at weeks 0 (baseline), week 12 and week 52 over the course of a GF diet being introduced/used. This included duodenal biopsies to assess the intensity of mucosal damage based on the Marsh criteria, a measure of intestinal permeability (IP) (yes, leaky gut) and various other tests, some of which are linked to the serology of CD (see here). Interestingly, vitamin D and vitamin B12 were also part of the testing suite.
• Various cognitive assessments were also included in the study covering aspects of memory, attention and language abilities, alongside a patient report measure on the presence of anxiety and depressive symptoms (something very topical at the moment).
• Results: bearing in mind this was a pilot study and involved only participants with CD all of whom were [knowingly] following a GF diet, there were some interesting points raised. The classical markers of CD saw their usual change as expected following use of a GF diet (compliance to which was described as excellent). The authors also note that: "IP was elevated in three participants, one of these at baseline the other two at week 12". Further: "All IP levels were within the normal range at 52 weeks".
• Based on the results of cognitive testing: "Four of the eight cognitive tests demonstrated a significant improvement in performance between time 0 and 52 weeks". These were predominantly tests in areas of attention, motor skills and verbal fluency. The authors note that this may be "the beginnings of an evidence-base for the ill-defined, yet frequently reported symptoms of brain fog in CD".
• The paper also reports some initial correlation values between some psychometric test scores and the CD histological and serological data. Obviously with 11 participants you would probably not imagine the correlation values, whether positive or negative, to be 'up there' but some of them were described as significant, and at better than just p<0.05.

Of course there is lots more to do in this area not least a larger and more controlled trial looking at the possibility of a relationship between CD, the GF diet and cognitive performance. The fact also that this was a study of adult CD also leaves questions to be answered about whether similar effects might be noticeable for paediatric cases of CD which appear to be on the rise. The other question resides in that murky grey area of non-coeliac gluten sensitivity (NCGS) and, assuming that this is a real phenomena (see here), whether a GF diet might also aid cognitive performance for cases there too? The paper by Volta and colleagues [2] (open-access) talks about a "foggy mind" as being apparent in some of their identified cases of NCGS...

This is not the first time that cognition has been talked about with CD in mind. "A possible association exists between progressive cognitive impairment and celiac disease, given the temporal relationship and the relatively high frequency of ataxia and peripheral neuropathy, more commonly associated with celiac disease". That was the conclusion of the study by Hu and colleagues [3]. Interestingly, they reported that out of a total of 13 participants, 3 people "improved or stabilized cognitively with gluten withdrawal". Terrone and colleagues [4] reported that in a paediatric cohort of kids recently diagnosed with CD, a measure of "cognitive, emotional and behavioural problems" was statistically higher than in another group of participant with CD symptoms controlled following the use of a GF diet. I was also quite interested to see that they reported a lower frequency of "chronic fatigue" in their CD remission group which brought back memories of a distant blog post I wrote on a similar topic (see here).

But the research is not yet cut-and-dried on the issue of cognitive dysfunction and untreated CD and any role for a GF diet. Take for example the small study by Hallert & Aström [5] (open-access here) who concluded "no consistent signs of cognitive impairment" to be found in their cohort. These results should also be take in light of other findings such as those by Casella and colleagues [6] looking at cognitive performance in older adults diagnosed with CD following a GF diet who concluded that: "Cognitive performance is worse in elderly coeliac disease than control patients, despite prolonged gluten avoidance in coeliacs". It's complicated.

Just before I leave you to further ponder the Lichtwark results, a few final points raised by the authors are also worthwhile mentioning. They speculate on the possible mechanisms by which "cognition might be impaired in patients with untreated CD". They do kinda rule out nutrient deficiencies on the basis of no association detected in their cohort with some of the nutritional parameters looked at. Personally, I don't think we can rule this out altogether at the moment given what we are still learning about good nutrition and behavioural and cognitive indicators (see here for one example). Inflammation and in particular, cytokine involvement receives a more favourable review to account for the results which should also be investigated further (see here). One final hypothesis revived by the authors is that of opioid peptides and any potential effects on "higher brain functions". I know brows become furrowed when the words 'opioid-excess' are talked about with reference to particular conditions, but given my own interest in this area, and recently our very preliminary paper on the formulation of low doses of the opiate blocker naltrexone into a cream [7] (see here), an intriguing question emerges about whether a GF diet might be the only avenue to enhancing cognitive performance under such circumstances? Oh and then there is the leaky gut angle too. At least one author [8] has asked the question of whether intestinal hyperpermeability might show some involvement in cognitive dysfunction albeit not necessarily directly pertinent to the issues of CD. More to do methinks.

To close, for those of you who might remember the film 'Limitless' and the fictional nootropic NZT-48, just think you could have it all. But also remember: "discontinued use will result in death". (And no, it's not real, not yet anyway).

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[1] Lichtwark IT. et al. Cognitive impairment in coeliac disease improves on a gluten-free diet and correlates with histological and serological indices of disease severity. Aliment Pharmacol Ther. 2014 May 28.

[2] Volta U. et al. An Italian prospective multicenter survey on patients suspected of having non-celiac gluten sensitivity. BMC Medicine 2014, 12:85.

[3] Hu WT. et al. Cognitive impairment and celiac disease. Arch Neurol. 2006 Oct;63(10):1440-6.

[4] Terrone G. et al. The Pediatric Symptom Checklist as screening tool for neurological and psychosocial problems in a paediatric cohort of patients with coeliac disease. Acta Paediatr. 2013 Jul;102(7):e325-8.

[5] Hallert C. & Aström J. Intellectual ability of adults after lifelong intestinal malabsorption due to coeliac disease. J Neurol Neurosurg Psychiatry. 1983 Jan;46(1):87-9.

[6] Casella S. et al. Cognitive performance is impaired in coeliac patients on gluten free diet: a case-control study in patients older than 65 years of age. Dig Liver Dis. 2012 Sep;44(9):729-35.

[7] Dodou K. et al. Ex vivo studies for the passive transdermal delivery of low-dose naltrexone from a cream; detection of naltrexone and its active metabolite, 6β-naltrexol, using a novel LC Q-ToF MS assay. Pharm Dev Technol. 2014 May 2.

[8] Brenner SR. Hypothesis: intestinal barrier permeability may contribute to cognitive dysfunction and dementia. Age Ageing (2010) 39 (2): 278-279.

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Lichtwark IT, Newnham ED, Robinson SR, Shepherd SJ, Hosking P, Gibson PR, & Yelland GW (2014). Cognitive impairment in coeliac disease improves on a gluten-free diet and correlates with histological and serological indices of disease severity. Alimentary pharmacology & therapeutics PMID: 24889390

Complementary and Alternative Medicine (CAM) use and autism

I want to talk today about the paper by Roger Akins and colleagues [1] and their finding that: "Use of CAM [Complementary and Alternative Medicine] is common in families of young children with neurodevelopmental disorders". I know that this topic has a tendency to furrow brows and roll eyes in some quarters, particularly when written or spoken alongside the word 'autism', so I'm treading carefully with this quite long post. I will also make reference to my oft-cited caveat: no medical or clinical advice is given or intended on this blog.

elixir vitae @ Chemical Heritage Foundation

CAM - not to be mistaken for CAMHS - covers an awful lot of ground when it comes to the range of 'treatment' options available; as the NHS Choices website puts it "treatments that fall outside of mainstream healthcare". Under the CAM umbrella you have a range of procedures and interventions which cover everything from nutrition to mind-body interventions and seemingly everything in between. Views on CAM - the various ideas behind it and it's perceived effectiveness in general - tend to be split as a function of lots of different variables.

I found quite a good overview looking at the main reasons why people tend to use CAM from a cancer care perspective (see here) which I assume can be similarly applied to CAM use for other conditions or ailments too. Specifically with autism in mind, I dare say that the issues of 'feeling more in control' and 'natural and healing therapies' (bearing in mind the continued mis-representation of the word 'chemical') would probably rank high on the list of why CAM enjoys a following. Indeed, other autism research seems to back up some of these assertions [2].

I've been aware of CAM with autism in mind pretty much from the beginning of my research career, and this is certainly not the first time that it's been talked about in the peer-reviewed domain [3] (open-access) with some pretty good reviews of what CAM covers autism-wise [4] (open-access). The fact that I embarked on a research life examining the potential effectiveness of a gluten- and casein-free (GFCF) diet as an intervention for some of the signs and symptoms associated with autism (and possibly some of it's various comorbidities) might even put me in the CAM researcher box. If the truth be known however, I've never been particularly happy with giving the GFCF diet a place under the CAM umbrella, simply because more and more we are seeing peer-reviewed evidence emerging suggestive that food does seem to be implicated in some cases of autism (remembering the 'not quite coeliac disease paper' for example) and, in a similar vein to the condition called PKU, how food may for some people, have effects that go well beyond just a nutritional perspective. Just sayin'.

Anyhow, the Akins paper, which has received quite a bit of media attention (see here and see here and see here), looked at data derived from the CHARGE initiative (beincharge!) which on this occasion asked parents of young children with autism (n=453) or developmental delay (DD) (n=125) what kinds of interventions or therapies had been used to try and affect the presentation of autism, both conventional and falling under the CAM label. They reported that roughly a third of children in both their autism and DD groups were or had used intervention(s) described as CAM.

Some of the most popular interventions reported were those related to nutrition; be that the use of vitamin and mineral supplements or the implementation of a GFCF diet (or combinations thereof). Indeed to quote from one of the media sources on this paper: "Nearly 7 percent of children with autism were on the gluten-free/casein-free diet, particularly children with frequent gastrointestinal problems". Interestingly these interventions alongside the use of things like probiotics were generally considered 'safe' by researchers. Several other interventions were also reported including vitamin B12 injections and the use of chelating agents which have also been talked about on this blog (see here and see here).

One of the first things to pass through my mind upon reading the Akins paper was the parent survey results from the Autism Research Institute (ARI) which asked parents 'what worked for their child with autism' (see here). I know, I know, the ARI document is not peer-reviewed science and subject to all manner of biases including that of potentially bringing out more positive than negative responses for specific interventions, but that was where the Akins paper led me and indeed, how the CAM use results seem to map on to the ARI results.

I personally think autism research can learn a few lessons from the Akins paper from several different perspectives. So:

• When setting the research agenda for autism in terms of where money and resources might be needed, further examination of the safety and efficacy of some of the interventions reported as being used might be a wise move [5]. As I've talked about before on this blog, there is some preliminary evidence that for example, dietary intervention (see here) or the use of vitamin and mineral supplements (see here) might be helpful for some people on the autism spectrum (or for some of the autisms if you prefer), but more study [methodologically stronger and replicative] is required. And some investigators have been trying to get funding for such research [6] for quite a while now...

• Going back to that suggestion that CAM might reflect more 'natural' therapies, I know there is some continued chatter about where CAM fits into the evidence-based medicine (EBM) model which we subscribe to these days. I don't want to get into the quagmire of EBM vs. the power of personal experience, but rather that further study is also required into the possible interactions that such CAM therapies may have with more conventional medicine. Quite a while back I talked about the interaction between fish oil supplements and chemotherapeutics (that is medication used in the treatment of cancer). What this and other studies point out is that your vitamin or nutritional supplement might be currently classified as a food supplement but that does not mean it can't have often quite potent biological effects. And certainly with the medicines cabinet seemingly being used for quite a few people on the autism spectrum (see here), there is scope for lots of potential interactions.

• It was interesting to read that Akins and colleagues examined various correlates which potentially might explain CAM use as opposed to non-CAM use. So, they report that "Families who used >20 [greater than] hours per week of conventional services were more likely to use CAM" which could indicate an issue of either degree of parent involvement or severity of autistic symptoms [7] in the decision to use CAM or not. Interesting too was the focus on "immunization status" as a variable examined with CAM use or non-use in mind and their finding that "Underimmunized children were marginally more likely to use CAM but not more likely to have received potentially unsafe or disproven CAM". I probably don't need to point this out, but this perhaps again might be further evidence for that 'natural vs. synthetic or man-made' issue potentially at the core of CAM use. It also illustrates one possibility why CAM use in autism still resides on the edge of mainstream autism research insofar as the generalised association often [incorrectly] made between certain types of CAM and the issue of vaccination (or rather 'anti-vaccination') [8]. I'm not saying that there isn't alignment between some purveyors of CAM and specific views on issues like vaccination [9], merely that not everyone holds those views and this should not be a barrier to further research on CAM. A little bit like what happens when talking about certain gastrointestinal (GI) issues in relation to autism and the way that conversation sometimes goes....

• The relationship between parents of children with autism and their healthcare providers is also touched upon in the Akins paper and how: "Further research should address how health care providers can support families in making decisions about CAM use". Huang and colleagues [10] recorded a potentially important point when it came to CAM use and autism in their study when indicating: "In children with ASD (the diagnostic group with the highest use of and satisfaction with CAM), physician's perceived knowledge of CAM was lower (14% versus 38%; p<0.05), as was perceptions of the physician's interest in learning more (p<0.05)." It is of course slightly unreasonable to expect every physician to know everything there is to know about CAM, particularly when applied to a condition as complex as autism. But given the age of the Internet and Dr. Google where all kinds of information is available to all, there is perhaps an even greater need for physicians to inform themselves about the ins-and-outs of such interventions and strategies and be able to advise accordingly based on the available evidence. Indeed, some physicians seem to be doing just that [11].

The Akins paper is certainly an interesting one in terms of highlighting how frequent CAM use is when it comes to autism. More than that though, it opens up a variety of discussion points [12] on the medical model, use of EBM, the patient-physician relationship and some of the complexities and politics of autism. That being said, we shouldn't lose sight of the fact that there are probably lots of reasons why parents of children with autism use or don't use CAM; probably in all cases, such decisions arrived at as a consequence of many very individual factors.

What the Akins paper did not address however was the important issue of how effective CAM use was and just as important, how ineffective CAM use was and for whom. This I assume being the next step in the research agenda...?

To close, readers in the UK will probably have heard about the death of Roger Lloyd Pack a.k.a Trigger from Only Fools and Horses. So, here's a theme tune which should bring back a memory of two... Dave.

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[1] Akins RO. et al. Utilization Patterns of Conventional and Complementary/Alternative Treatments in Children with Autism Spectrum Disorders and Developmental Disabilities in a Population-Based Study. J Dev Behav Ped. 2014; 35: 1-10.

[2] Hanson E. et al. Use of complementary and alternative medicine among children diagnosed with autism spectrum disorder. J Autism Dev Disord. 2007 Apr;37(4):628-36.

[3] Levy SE. & Hyman SL. Complementary and Alternative Medicine Treatments for Children with Autism Spectrum Disorders. Child Adolesc Psychiatr Clin N Am. 2008 October; 17(4): 803–ix.

[4] Lofthouse N. et al. A Review of Complementary and Alternative Treatments for Autism Spectrum Disorders. Autism Res Treatment. 2012; 870391.

[5] Anagnostou E. & Hansen R. Medical treatment overview: traditional and novel psycho-pharmacological and complementary and alternative medications. Curr Opin Pediatr. 2011 Dec;23(6):621-7.

[6] Winburn E. et al. Parents' and Child Health Professionals' Attitudes Towards Dietary Interventions for Children with Autism Spectrum Disorders. J Autism Dev Disord. 2013 Sep 1.

[7] Perrin JM. et al. Complementary and alternative medicine use in a large pediatric autism sample. Pediatrics. 2012 Nov;130 Suppl 2:S77-82.

[8] Gupta VB. Communicating with parents of children with autism about vaccines and complementary and alternative approaches. J Dev Behav Pediatr. 2010 May;31(4):343-5.

[9] Ernst E. Rise in popularity of complementary and alternative medicine: reasons and consequences for vaccination. Vaccine. 2001 Oct 15;20 Suppl 1:S90-3

[10] Huang A. et al. Parental perspectives on use, benefits, and physician knowledge of complementary and alternative medicine in children with autistic disorder and attention-deficit/hyperactivity disorder. J Altern Complement Med. 2013 Sep;19(9):746-50.

[11] Golnik AE. & Ireland M. Complementary alternative medicine for children with autism: a physician survey. J Autism Dev Disord. 2009 Jul;39(7):996-1005.

[12] Committee on Children with Disabilities. American Academy of Pediatrics: Counseling families who choose complementary and alternative medicine for their child with chronic illness or disability. Committee on Children With Disabilities. Pediatrics. 2001 Mar;107(3):598-601.

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Akins, Roger S., Krakowiak, Paula, Angkustsiri, Kathleen, Hertz-Picciotto, Irva, & Hansen, Robin L. (2014). Utilization Patterns of Conventional and Complementary/Alternative Treatments in Children with Autism Spectrum Disorders and Developmental Disabilities in a Population-Based Study Journal of Developmental & Behavioral Pediatrics : http://journals.lww.com/jrnldbp/Abstract/2014/01000/Utilization_Patterns_of_Conventional_and.1.aspx

Methylcobalamin and folinic acid for autism? Hold it right there...

The title of this post should also have included the word 'glutathione' too based on the results reported by Richard Frye and colleagues* (open-access) describing behavioural and biochemical data from a 3-month open trial of methylcobalamin, a vitamin B12 'vitamer', and folinic acid with a group of children diagnosed with an autism spectrum disorder (ASD).

Dr Frye and his various research are no stranger to this blog; ranging from mitochondrial dysfunction linked to cases of autism (see here), through to tetrahydrobiopterin (BH4) and autism (see here), and folate receptor autoantibodies and autism (see here). Indeed that last link on folate receptor autoantibodies brings into view a potential reason why folinic acid was included in their recent study. That and the inclusion of Jill James on the authorship list and her previous studies looking at combined methylcobalamin and folinic acid supplementation for autism** (open-access) discussed as part of a previous post (see here).

The crux of this study was the suggestion that in the great and complex pathway which links the recycling of homocysteine (the big 'H') and the folate cycle, there is potentially enough going on in cases of autism to interfere with (a) the process of methylation (see here) and (b) that most useful of compounds, glutathione (see here), well reduced glutathione anyway. I'm also inclined to point readers the way of the very thorough analysis of glutathione and autism produced by Main and colleagues*** (open-access) a while back (see here) just in case you think I'm talking biochemical mumbo-jumbo.

Readers might already have seen mention of the words 'open trial' at the top of this post. This indicating that the latest study from Frye and colleagues was a case of following 37 children who fitted the entrance criteria - including "abnormal methylation capacity (SAM/SAH < 3.0) and glutathione redox metabolism (GSH/GSSG < 6.0)" - and seeing how they went over the course of a "sterile subcutaneous injection of methylcobalamin in the fatty tissue of the buttocks" every 3 days combined with oral delivery of folinic acid twice daily mixed with food. For those wincing or furrowing their brows about those injections of methylcobalamin with children with autism, I'll just say that the issue of drug delivery has been talked about in a previous post and this study was passed through an ethics committee "at the University of Arkansas for Medical Sciences".

The results are interesting. Quite a few behavioural changes were documented according to use of the VABS. This bearing in mind that (a) there was no control or placebo group and (b) VABS is a parent-report schedule which in this case merely looked at unblinded pre- and post-intervention scores. Nonetheless, the intervention resulted in "significant increases in VABS scores for all domains, including daily living, social, and communication skills, with an average effect size of 0.59, which is in the medium-to-large range." The authors even went as far to say that the VABS changes indicated something like an average 7.7 month gain over the 3 month period of study.

All well and good with that open-trial caveat well and truly in place. It is however the details regarding the biochemical measure of glutathione measurement that I was more interested in. Indeed, if I had to suggest one improvement to this paper, it would have been to include a simple table showing glutathione measures - GSH/GSSG - at baseline compared with at 3 months. Instead, the glutathione results are all bundled up with the VABS results as per the example of figure 3 showing: "the change in the glutathione redox status (reduced-to-oxidized glutathione ratio) and change in subscales of the Vineland Adaptive Behavior Scale (VABS) subscales".

What I did manage to glean is that: "the overall glutathione redox status was not related to VABS subscales, indicating that overall development did not appear to be related to overall glutathione redox status". Fair enough, a possible selection issue based on the group eventually included for study. It was the change in glutathione redox status after intervention which seemed to tie into the VABS results reported. Still, I would have liked to have the biochemical data presented as a stand-alone table.

I'm trying not to be overly-critical of this paper and results contained within. As with many other researchers, I'm guilty of the odd open-trial forming part of my CV (see here). Whilst useful as a starting point for looking at a particular intervention or trying to get others to do a more methodologically-sound study, one has to be quite cautious of such work and the myriad of biases that they contain.

I do get the impression that outside of just a more methodologically-sound trial, a lot more questions need to be asked about this intervention regime before it can be considered as something more mainstream. Outside of the 2 children who dropped out of the study because "parents were uncomfortable giving the methylcobalamin injections", there's also a question of what such an intervention is actually doing. I note the authors when discussing the previous James trial**, are quoted as saying: "The fact that the treatment [methylcobalamin and folinic acid] improved but did not normalize methionine, SAM and glutathione concentrations may reflect ongoing metabolic compensation for incompletely resolved oxidative stress". This may very well be true, but could also indicate that intervention was also working on other biological systems too.

That also mention is made of the Hardan trial of N-acetlycysteine (NAC) for autism (see here) and NAC being a direct glutathione precursor, suggests to me that when it comes to glutathione production, the shortest point might be A to B bearing in mind what results have been obtained from direct glutathione supplementation**** (open-access).

To close, the Clash have a question for you.... (it's the indecisions which bug me).

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* Frye RE. et al. Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status. Autism Res Treat. 2013: 609705.

** James SJ. et al. Efficacy of methylcobalamin and folinic acid treatment on glutathione redox status in children with autism. Am J Clin Nutr. 2009 January; 89(1): 425–430.

*** Main PA. et al. The potential role of the antioxidant and detoxification properties of glutathione in autism spectrum disorders: a systematic review and meta-analysis. Nutr Metab (Lond). 2012 Apr 24;9:35.

**** Kern JK. et al. A clinical trial of glutathione supplementation in autism spectrum disorders. Med Sci Monit. 2011 Dec;17(12):CR677-82.

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Richard Frye, Stepan Melnyk, George Fuchs, Tyra Reid, Stefanie Jernigan, Oleksandra Pavliv, Amanda Hubanks, David W. Gaylor, Laura Walters, S. Jill James (2013). Effectiveness of Methylcobalamin and Folinic Acid Treatment on Adaptive Behavior in Children with Autistic Disorder Is Related to Glutathione Redox Status Autism Research and Treatment DOI: 10.1155/2013/609705